QUALITATIVE TESTS FOR CARBOHYDRATES Page 1 of 8

Qualitative Tests for Carbohydrates

Potato consists of different carbohydrates like starch, reducing sugars
etc. Difficulties are encountered in the qualitative and quantitative
analysis of samples containing mixtures of carbohydrates, particularly
the sugars, because of their structural and chemical similarity and also
with respect to their stereoisomers. During biochemical investigations it
may because necessary to establish whether a given sample,
particularly of a purified preparation, consist carbohydrates or not.
Several rapid tests are available the presence or absence of a sugar or
a carbohydrate in a sample. These tests are based on specific colour
reactions typical for their group and are described below. For laboratory
practical, it may be advised to perform these tests with the individual
rather than mixture of sugars. Use of sugar solutions of different
concentrations (0.1-1%) during these experiments would also provide
valuable information about the sensitivity of these tests. The types of
carbohydrates detected by these tests are:

Name of the test Application

1. Molisch’s Test General test for carbohydrates

2. Anthone Test General test for carbohydrates

3. Iodine Test For glycans (starch, glycogen)

4. Barfoed’s Test To distinguish between mono-saccharides from

reducing diasaccharides

5. Seliwanoff’s Test For Ketones

6. Fehling’s Test For reducing sugars

7. Bendict’s Test For reducing sugars

8. Picric acid Test For reducing sugars

9. Bial’s Test For pentoses

1) MOLISCH’S TEST

Principle

This is a general test for all carbohydrates. Conc. H2SO4

hydrates glycosidic bonds to yield monosaccharides which in
the presence of an acid get dehydrated to form furfural and its
derivatives. These products react with sulphonated α-naphthol
to give a purple complex. Polysaccharides and glycoproteins
also give a positive reaction.

Reaction

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Reagents

1. Conc. H2SO4

2. α-naphthol: 5% (w/v) in ethanol (prepare fresh)

Procedure and observations

Add 2-3 drops of α-naphthol solution to 2 ml of the test

solution. Very gently pipette 1ml conc. H2SO4 along the side if
the test tube so that the two distinct layers are formed.
Carefully observe any color change at the junction two layers.
Appearance of purpose color indicates the presence of
carbohydrates in the sample preparation or the test solution.

Precautions

1. α-naphthol solution is unstable and should be prepared fresh.

2. Conc. H2SO4 should be along the sides of the test tubes causing
minimal disturbance to the contents in the tube.

2) ANTHRONE TEST

Principle

Anthrone reaction is another general test for carbohydrates. In

this the furfural produced reacts with anthrone to give bluish
green colored complex.

Reaction

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Materials and Reagents

1. Boiling water bath.

2. Conc. H2SO4
3. 0.2% (w/v) anthrone solution

Procedure and observations

Add 0.5 - 1 ml of the test solution to about 2 ml of anthrone

reagent and mix thoroughly. Observe whether the color
changes to bluish green. If not, examine the tubes again
keeping them in boiling water bath for 10 min.

3) IODINE TEST

Principle

Iodine forms colored adsorption complexes with

polysacchaides. Starch gives blue color with iodine, while
glycogen reacts to form reddish brown complex. Hence it is
useful, convenient and rapid test for detection of amylase,
amylopectin and glycogen.

Reagents

1. Iodine solution: Prepare 0.005N iodine solution in 3% (w/v)

potassium iodine solution.
2. 1% Test solutions of glucose, sucrose, starch, glycogen, cellulose
etc.

Procedure and observations

Take 1 ml of the sample extract or test solution in a test tube.

Add 4 - 5 drops of iodine solution to it and mix the contents
gently. Observe if any colored product is formed. Note the
color of the product.

4) Barfoed’s Test

Principle

This test is used for distinguishing monosaccharides from

reducing disaccharides. Monosaccharides usually react in

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about 1 - 2 min while the reducing disaccharides take much

longer time between 7 - 12 min to get hydrolysed and then
react with the reagent. Brick red color is obtained in this test
which is due to the formation of cuprous oxide.

Reaction

(CH3COO)2Cu2 + H2O ® 2CH3COOH + Cu(OH)2

Cupric acetate Cupric hydroxide

Cu(OH)2 ® CuO+H2O

Materials and Reagents

1. Boiling water bath

2. Barfoed’s reagents: Dissolve 13.3 g of copper acetate in 200 ml
water and add 1.8 ml of glacial acetic acid to it.

Procedure and observations

Take 2 ml of Barfoed’s solution in a test tube and add 1ml of

sample solution to it. Keep the test tubes in a boiling water
bath. A briskly boiling water bath should be used for obtaining
reliable results. Look for the formation of brick red color and
also note the time taken for its appearance.

5) SELIWANOFF’S TEST

Principle

This test is used to distinguish aldoses from ketoses. Ketoses

undergo dehydration to give furfural derivatives, which then
condense with resorcinol to form a red complex. Prolonged
heating will hydrolyze disaccharides and other
monosaccharides will also eventually give color.

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Reaction

Materials and Reagents

1. Boiling water bath

2. Seliwanoff’s reagent: 0.05% (w/v) resorcinol in 3 HCl

Procedure and observations

Add 1ml of the test solution to 2 ml of Seliwanoff’s reagent

and warm in a boiling water bath for 1min. Note for the
appearance of a deep red color. This would indicate that the
sample solution contains a keto sugar.

6) Fehling’s Test

Principle

Fehling’s test is a specific and highly sensitive for detection of

reducing sugars. Formation of yellow or red ppt of cuprous
oxide denotes the presence of reducing sugars. Rochelle salt
acts as the chelating agent in this reaction.

Reaction

Materials and Reagents

1. Boiling water bath.

2. Fehling’s solution A: Dissolve 35 g of CuSO4.5H2O in water and
make the volume to 500 ml.
3. Fehling’s solution B: Dissolve 120 g of KOH and 173 g Na-K
tartrate (Rochelle salt) in water and make the volume to 500 ml.

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4. Fehling’s reagent: Mix equal volumes of Fehling’s solution A and B.

These solutions must be mixed immediately prior to use.

Procedure and observations

Add 1 ml of Fehling’s reagent (Reagent No. 4) to 1 ml of

aliquot of the test solution. Mix thoroughly and place the test
tubes in vigorously boiling water bath. Look out for the
formation of red ppt of cuprous oxide which would indicate the
presence of reducing sugars in the solution.

7) Benedict’s test

Principle

Benedict’s test is more convenient and this reagent in more

stable. In this method sodium citrate functions as a chelating
agent. Presence of reducing sugars results in the formation of
red ppt of cuprous oxide.

Reaction

Materials and Reagents

1. Boiling water bath.

2. Benedict’s reagents: Dissolve 173 g of sodium citrate and 100 g of
anhydrous Na2CO3 in 600 ml of hot H2O. Dilute to 800 ml with
water.
3. Dissolve 17.3 g of CuSO4.H2O in 100 ml hot water. Cool and dilute
to 100 ml.
4. Add Reagent No.2 to Reagent No.3 slowly with constant stirring.
Make the final volume to 1 L.

Procedure and observations

Add 0.5 - 1 ml of the test solution or sample extract to 2 ml of

Benedict’s reagent (Reagent No. 4). Keep the test tubes in a
vigorously boiling water bath. Observe for the formation of red
precipitates whose appearance would suggest the presence
of reducing sugars in the given or sample extract.

8) Picric acid test

Principle

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It is another test for detection of reducing sugars. The

reducing sugars react with picric acid to form a red colored
picramic acid.

Reaction

Materials and Reagents

1. Boiling water bath.

2. Saturated picric acid: Dissolve 13 g picric acid in distilled water,
boil and cool.
3. 10% Na2CO3.

Procedure and observations

Add 1 ml saturated picric acid to 1 ml of sample solution

followed by 0.5 ml 10% Na2CO3. Heat the test tubes in a
boiling water bath. Appearance of red color would indicate the
presence of reducing sugars in the sample solution.

9) Bial’s test

Principle

This test is useful in the determination of pentose sugars.

Reaction is due to formation of furfural in the acid medium
which condenses with orcinol in presence of ferric ions to give
a blue-green colored complex which is soluble in butyl
alcohol.

Reaction

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Materials and Reagents

1. Boiling water bath

2. Dissolve 1.5 g of orcinol in 100 ml of conc. HCl and add 20-30
drops of 10 % ferric chloride solution to it.

Procedure and observations

To 2 ml of Bial’s reagent add 4-5 drops of test solution and

heat in a boiling water bath. Observe for the formation of blue-
green colored complex.

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